Peripheral vascular occlusive disease (PVOD) is a major cause of patient morbidity and mortality is the USA, leading to 150,000 amputations per year (1). In spite of the recent advances in the management of PVOD, many patients with critical limb ischemia are not candidates for surgical revascularization due to the lack of an appropriate conduit or adequate outflow artery required for successful and durable bypass procedures. For these patients, limb amputation is the only option. An alternate approach would be to use gene therapy to promote angiogenesis to improve blood flow rate to critically ischemic skeletal muscle, thereby preventing limb loss and hopefully improving the quality of life for the patient (2). Recently, gene therapy directed at over expression of vascular endothelial growth factor (VEGF) has emerged as a potential technique to promote limb angiogenesis. VEGF is expressed selectively by endothelial cells and is upregulated by hypoxia (3, 4). Endothelial nitric oxide synthase (eNOS) is thought to be a downstream effector of VEGF (5, 6). Preliminary data from Dr Messina's laboratory suggests that over expression of eNOS in a rat hindlimb ischemia model significantly increases tissue oxygen concentration as well as blood flow rate. The overall goal of this proposal is to optimize the eNOS mediated increases in tissue oxygen concentration and blood flow rate. This goal leads to these specific aims: 1) Document the effect of adenoviral- wediated gene transfer of eNOS to an ischemic hindlimb on skeletal muscle viability, tissue oxygen concentration, blood flow rate, and an overall clinical ischemic index of the limb. 2) Optimize the beneficial effect of eNOS over expression to reverse critical limb ischemia. 3) Document the durability of the effect of transient eNOS over expression on skeletal muscle viability, tissue oxygen concentration, blood flow rate, and overall clinical ischemic index of the limb.